The broad, long-term objectives of this application are development and application of new single molecule methodologies for ultra-rapid, high resolution human genomic analysis. Mapping rates and resolution can be enormously improved if new molecular sizing methodologies are developed and fully integrated into the current mapping efforts. To this end, Optical Mapping was developed in this laboratory, which is a powerful non- electrophoretic approach to rapidly create high-resolution ordered maps from single eucaryotiC chromosomal DNA molecules. Optical Mapping rapidly produces ordered maps by imaging single DNA molecules using fluorescence microscopy during restriction enzyme digestion. Resulting fragments are sized by relative intensity or relative apparent length. Development is proceeding on a series of newer single molecule sizing methodologies which use molecular relaxation phenomena to produce a high degree of size discrimination. Also, advanced fluorescence intensity measurement techniques are being developed to bring size resolution to the 500 base level. Optical Mapping can produce maps from almost any source of DNA, including YACs and genomic DNA. Use of genomic DNA will enable the widespread clinical usage of genomic mapping data since construction of high resolution maps will be possible for individuals. Sequence-specific regions of large naked DNA molecules can be precisely mapped using a combination of Optical Mapping with a published technique (RARE), which is a combination of RecA protein mediated hybridization, methylation, and endonuclease cleavage. Further modification of RecA-based approaches are designed to increase its sensitivity and specificity by the combination of various single molecule tagging techniques. Energy transfer techniques will be used to increase sensitivity and specificity of detection of these tags. Finally, the adaptation of simple flow procedures will increase the throughput of Optical Mapping and greatly facilitate high resolution mapping and ordering of large genomic regions, YACs or contigs that will serve both scientific and clinical needs.